Use of peptide epoxyketones for metastasis suppression

ABSTRACT

The invention provides a method of repressing metastasis of a cancer compromising the administration of a peptide epoxyketone proteasome inhibitor. Furthermore, the method can be performed in combination with the administration of one or more additional therapeutics.

This application claims priority to U.S. Application No. 61/261,062,filed on Nov. 13, 2009, with the United States Patent and TrademarkOffice, the entire contents of which applications are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Metastasis is the spread of a disease from one organ or part to anothernon-adjacent organ or part. It is commonly associated with cancer, wherecancer cells migrate from the place where the cancer started (the“primary tumor”) to other parts of the body to form a new tumor. The newtumor is a “metastic” or “secondary” tumor of the primary tumor. Thus,if breast cancer cells metastasize to the lungs, the secondary tumor iscalled metastatic breast cancer, not lung cancer. Most tumors and otherneoplasms can metastasize; thus, the clinical management of metastasisis of great importance.

An extraordinarily complex process, metastasis consists of a series ofimportant steps. These steps include detachment of tumor cells from theprimary tumor, invasion through surrounding tissues and basementmembranes, entry and survival in the circulation, lymphatic system orperitoneal space, and establishment and proliferation of the tumor cellsin a distant target organ. The specific molecular mechanisms behindthese steps remain unclear, thus, identifying effective methods toprevent or suppress tumor metastasis has been challenging. New methodsare needed.

SUMMARY OF THE INVENTION

While having a general function in intracellular protein turnover, theproteasome controls the levels of proteins that are important forcell-cycle progression and apoptosis in normal and malignant cells, forexample, cyclins, caspases, BCL-2 and nF-kB (Kumatori et al., Proc.Natl. Acad. Sci. USA (1990) 87:7071-7075; Almond et al., Leukemia (2002)16: 433-443). Many of these proteins are key components in the stepsinvolved in the metastatic process. Therefore, inhibiting proteasomeactivity can translate into therapies to treat various disease states,such as the prevention or repression of metastic tumors.

In certain embodiments, the present invention relates to methods forrepressing or preventing the metastatic spread of cancer, comprisingadministering a peptide epoxyketone proteasome inhibitor. In certainsuch embodiments, the peptide epoxyketone proteasome inhibitor is atripeptide epoxyketone.

The peptide epoxyketone may be administered by any of various modes. Incertain embodiments, the peptide epoxyketone is administered orally.

In certain embodiments of the present invention, the peptide epoxyketoneproteasome inhibitor may be used in combination with other therapeuticagents. The additional therapeutics may be known at the time of thisapplication, or may become apparent after the date of this application.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a reduction in the number of metastatic breast tumors withthe administration of Compound 1.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, the invention relates to a method to repress orprevent the metastasis of a cancer in an individual, comprisingadministering a peptide epoxy ketone. In some embodiments, theindividual is a mammal. In certain preferred embodiments the individualis a human.

In certain embodiments, the peptide epoxyketone has a structure offormula (I) or a pharmaceutically acceptable salt thereof,

wherein

-   each Ar is independently an aromatic or heteroaromatic group    optionally substituted with 1 to 4 substituents;-   L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ or    C═O;-   X is selected from O, S, NH, and N—C₁₋₆alkyl, preferably 0;-   Y is absent or is selected from C═O and SO₂;-   Z is absent or is C₁₋₆alkyl;-   R¹, R², and R³ are each independently selected from C₁₋₆alkyl,    C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, any of    which is optionally substituted;-   R⁴ is N(R⁵)L-Z—R⁶;-   R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl-Y—, and C₁₋₆alkyl-Y—,    preferably hydrogen;-   R⁶ is selected from hydrogen, OR⁷, C₁₋₆alkenyl, Ar—Y—, carbocyclyl,    and heterocyclyl; and-   R⁷ and R⁸ are independently selected from hydrogen, C₁₋₆alkyl, and    C₁₋₆aralkyl, preferably hydrogen.

In certain embodiments, L is selected from C═O, C═S, and SO₂, preferablySO₂ or C═O.

In certain embodiments, R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl,and C₁₋₆alkyl, preferably hydrogen.

In certain embodiments, R⁶ is selected from hydrogen, C₁₋₆alkenyl,Ar—Y—, carbocyclyl, and heterocyclyl.

In certain embodiments, X is O and R¹, R², and R³ are each independentlyselected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl. In preferredsuch embodiments, R¹ and R³ are independently C₁₋₆alkyl and R² isC₁₋₆aralkyl. In more preferred such embodiments, R¹ and R³ are bothisobutyl and R² is phenylmethyl.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, R⁶ is Ar—Y—,and each Ar is independently selected from phenyl, indolyl,benzofuranyl, naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl,pyrazyl, and the like. In certain such embodiments, Ar may besubstituted with Ar-Q-, where Q is selected from a direct bond, —O—, andC₁₋₆alkyl. In certain other such embodiments where Z is C₁₋₆alkyl, Z maybe substituted, preferably with Ar, e.g., phenyl.

In certain embodiments, R⁵ is hydrogen, Z is absent, L is C═O or SO₂,and R⁶ is selected from Ar—Y and heterocyclyl. In certain preferred suchembodiments, heterocyclyl is selected from chromonyl, chromanyl,morpholino, and piperidinyl. In certain other preferred suchembodiments, Ar is selected from phenyl, indolyl, benzofuranyl,naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl, pyrazyl, and thelike.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, Z is absent,and R⁶ is C₁₋₆alkenyl, where C₁₋₆alkenyl is a substituted vinyl groupwhere the substituent is preferably an aryl or heteroaryl group, morepreferably a phenyl group optionally substituted with one to foursubstituents.

In certain embodiments, R⁷ and R⁸ are independently selected fromhydrogen and C₁₋₆alkyl. In certain preferred such embodiments, R⁷ and R⁸are independently selected from hydrogen and methyl. In more preferredsuch embodiments, R⁷ and R⁸ are both hydrogen.

In certain alternative embodiments, the peptide epoxyketone has astructure of Formula (II) or a pharmaceutically acceptable salt thereof:

wherein

L is selected from C═O, C═S, and SO₂, preferably C═O;

X is O;

Z is absent, C₁₋₆alkyl, or C₁₋₆alkoxy, preferably absent;

R¹, R², and R³ are each independently selected from hydrogen, C₁₋₆alkyl,C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl,C₁₋₆aralkyl, heteroaryl, heterocyclyl, C₁₋₆heterocycloalkyl,C₁₋₆heteroaralkyl, carbocyclyl, and C₁₋₆carbocyclolalkyl;

R⁴ is selected from hydrogen, C₁₋₆aralkyl, and C₁₋₆alkyl;

R⁵ is heteroaryl; and

R⁶ and R⁷ are independently selected from hydrogen, C₁₋₆alkyl, andC₁₋₆aralkyl.

In certain embodiments, R¹, R², and R³ are independently selected fromhydrogen, C₁₋₆ alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, C₁₋₆aralkyl,C₁₋₆heterocycloalkyl, C₁₋₆heteroaralkyl, and C₁₋₆carbocyclolalkyl. Incertain embodiments, any of R¹, R², and R³ are independently C₁₋₆alkylselected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, andisobutyl. In certain embodiments, any of R¹, R², and R³ areindependently C₁₋₆hydroxyalkyl. In certain preferred such embodiments,any of R¹, R², and R³ are independently selected from hydroxymethyl andhydroxyethyl, preferably hydroxymethyl. In certain embodiments, any ofR¹, R², and R³ are independently C₁₋₆alkoxyalkyl. In certain suchembodiments, any of R¹, R², and R³ are independently selected frommethoxymethyl and methoxyethyl, preferably methoxymethyl. In certainembodiments, any of R¹, R², and R³ are independently C₁₋₆heteroaralkyl.In certain such embodiments, any of R¹, R², and R³ are independentlyselected from imidazolylmethyl, pyrazolylmethyl, and thiazolylmethyl,and pyridylmethyl, preferably imidazol-4-ylmethyl, thiazol-4-ylmethyl,2-pyridylmethyl, 3-pyridylmethyl, or 4-pyridylmethyl. In certainembodiments, any of R¹, R², and R³ are independently C₁₋₆aralkyl. Incertain such embodiments, any of R¹, R², and R³ are independentlyselected from phenylmethyl(benzyl) and phenylethyl, preferablyphenylmethyl. In certain embodiments, any of R¹, R², and R³ areindependently C₁₋₆carbocycloalkyl. In certain such embodiments, R¹ iscyclohexylmethyl. In certain embodiments R¹, R², and R³ are alldifferent. In certain embodiments, any two of R¹, R², and R³ are thesame. In certain embodiments, R¹, R², and R³ are all the same.

In certain embodiments, at least one of R¹ and R² is selected fromC₁₋₆hydroxyalkyl and C₁₋₆alkoxyalkyl. In certain such embodiments, atleast one of R¹ and R² is alkoxyalkyl. In certain such embodiments, atleast one of R¹ and R² is selected from methoxymethyl and methoxyethyl.

In certain embodiments, R³ is selected from C₁₋₆alkyl and C₁₋₆aralkyl,preferably C₁₋₆alkyl. In certain such embodiments, R³ is selected frommethyl, ethyl, isopropyl, sec-butyl, and isobutyl. In certain suchembodiments, R³ is isobutyl. In certain alternative embodiments, R³ isselected from phenylmethyl and phenylethyl, preferably phenylmethyl.

In certain embodiments, R⁴, R⁶, and R⁷ are independently selected fromhydrogen and methyl, preferably hydrogen.

In certain embodiments, R⁵ is a 5- or 6-membered heteroaryl. In certainsuch embodiments, R⁵ is selected from isoxazole, isothiazole, furan,thiophene, oxazole, thiazole, pyrazole, or imidazole, preferablyisoxazole, furan, or thiazole.

In certain embodiments, R⁵ is a bicyclic heteroaryl. In certain suchembodiments, bicyclic heteroaryl is selected from benzisoxazole,benzoxazole, benzothiazole, benzisothiazole.

In certain embodiments, L is C═O, Z is absent, and R⁵ is anisoxazol-3-yl or isoxazol-5-yl. In certain preferred such embodiments,when the isoxazol-3-yl is substituted, it is substituted at least at the5-position. In certain preferred embodiments, when the isoxazol-5-yl issubstituted, it is substituted at least at the 3-position.

In certain embodiments, L is C═O, Z is absent, and R⁵ is anunsubstituted isoxazol-3-yl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is a substitutedisoxazol-3-yl. In certain such embodiments, R⁵ is isoxazol-3-ylsubstituted with a substituent selected from C₁₋₆alkyl, C₁₋₆alkoxy,C₁₋₆alkoxyalkyl, C₁₋₆hydroxyalkyl, carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂aminocarboxylate,C₁₋₆alkylcarboxylate, C₁₋₆heteroaralkyl, C₁₋₆aralkyl,C₁₋₆heterocycloalkyl, and C₁₋₆carbocycloalkyl. In certain preferred suchembodiments, R⁵ is isoxazole-3-yl substituted with a substituentselected from methyl, ethyl, isopropyl, and cyclopropylmethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with a 4- to 6-membered nitrogen-containingC₁₋₆heterocycloalkyl. In certain such embodiments, R⁵ is isoxazol-3-ylsubstituted with azetidinylmethyl, preferably azetidin-1-ylmethyl. Incertain alternative such embodiments, L is C═O, Z is absent, and R⁵ isisoxazol-3-yl substituted with

wherein W is O, NR, or CH₂, and R is H or C₁₋₆alkyl. In certain suchembodiments, W is O.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with 5-membered nitrogen-containing C₁₋₆heteroaralkyl, suchas pyrazolylmethyl, imidazolylmethyl, triazol-5-ylmethyl, preferably1,2,4-triazol-5-ylmethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with C₁₋₆alkoxy or C₁₋₆alkoxyalkyl, preferably methoxy,ethoxy, methoxymethyl, or methoxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with C₁₋₆hydroxyalkyl, preferably hydroxymethyl orhydroxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with a carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂aminocarboxylate, orC₁₋₆alkylcarboxylate. In certain such embodiments, R⁵ is substitutedwith methyl carboxylate or ethyl carboxylate, preferably methylcarboxylate.

In certain embodiments, L is C═O, Z is absent, and R⁵ is anunsubstituted isoxazol-5-yl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is a substitutedisoxazol-5-yl. In certain such embodiments, R⁵ is isoxazol-5-ylsubstituted with a substituent selected from C₁₋₆alkyl, C₁₋₆alkoxy,C₁₋₆alkoxyalkyl, C₁₋₆hydroxyalkyl, carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂aminocarboxylate,C₁₋₆alkylcarboxylate, C₁₋₆heteroaralkyl, C₁₋₆aralkyl,C₁₋₆heterocycloalkyl, and C₁₋₆carbocycloalkyl. In certain preferred suchembodiments, R⁵ is isoxazole-3-yl substituted with a substituentselected from methyl, ethyl, isopropyl, and cyclopropylmethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with a 4- to 6-membered nitrogen-containingC₁₋₆heterocycloalkyl. In certain such embodiments, R⁵ is isoxazol-5-ylsubstituted with azetidinylmethyl, preferably azetidin-1-ylmethyl. Incertain alternative such embodiments, L is C═O, Z is absent, and R⁵ isisoxazol-3-yl substituted with

wherein W is O, NR, or CH₂, and R is H or C₁₋₆alkyl. In certain suchembodiments, W is O.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-5-ylsubstituted with 5-membered nitrogen-containing C₁₋₆heteroaralkyl, suchas pyrazolylmethyl, imidazolylmethyl, triazol-5-ylmethyl, preferably1,2,4-triazol-5-ylmethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-5-ylsubstituted with C₁₋₆alkoxy or C₁₋₆alkoxyalkyl, preferably methoxy,ethoxy, methoxymethyl, or methoxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-5-ylsubstituted with C₁₋₆hydroxyalkyl, preferably hydroxymethyl orhydroxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with a carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂aminocarboxylate, orC₁₋₆alkylcarboxylate. In certain such embodiments, R⁵ is substitutedwith methyl carboxylate or ethyl carboxylate, preferably methylcarboxylate.

In certain preferred embodiments, a compound of Formula (II) has astructure

DEFINITIONS

The term “C_(x-y)alkyl” refers to substituted or unsubstituted saturatedhydrocarbon groups, including straight-chain alkyl and branched-chainalkyl groups that contain from x to y carbons in the chain, includinghaloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.C₀alkyl indicates a hydrogen where the group is in a terminal position,a bond if internal. The terms “C_(2-y)alkenyl” and “C_(2-y)alkynyl”refer to substituted or unsubstituted unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxy.

The term “C₁₋₆alkoxyalkyl” refers to a C₁₋₆alkyl group substituted withan alkoxy group, thereby forming an ether.

The term “C₁₋₆aralkyl”, as used herein, refers to a C₁₋₆alkyl groupsubstituted with an aryl group.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by the general formulae:

wherein R⁹, R¹⁰ and R^(10′) each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R⁸, or R⁹ and R¹⁰ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R⁸ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger from 1 to 8. In preferred embodiments, only one of R⁹ or R¹⁰ canbe a carbonyl, e.g., R⁹, R¹⁰, and the nitrogen together do not form animide. In even more preferred embodiments, R⁹ and R¹° (and optionallyR^(10′)) each independently represent a hydrogen, an alkyl, an alkenyl,or —(CH₂)_(m)—R⁸. In certain embodiments, the amino group is basic,meaning the protonated form has a pK_(a)≧7.00.

The terms “amide” and “amido” are art-recognized as an amino-substitutedcarbonyl and includes a moiety that can be represented by the generalformula:

wherein R⁹, R¹⁰ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsubstituted or unsubstituted single-ring aromatic groups in which eachatom of the ring is carbon. The term “aryl” also includes polycyclicring systems having two or more cyclic rings in which two or morecarbons are common to two adjoining rings wherein at least one of therings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline,and the like.

The terms “carbocycle” and “carbocyclyl”, as used herein, refer to anon-aromatic substituted or unsubstituted ring in which each atom of thering is carbon. The terms “carbocycle” and “carbocyclyl” also includepolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is carbocyclic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R¹¹represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R⁸ or apharmaceutically acceptable salt, R^(11′) represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R⁸, where m and R⁸ are as defined above.Where X is an oxygen and R¹¹ or R^(11′) is not hydrogen, the formularepresents an “ester”. Where X is an oxygen, and R¹¹ is a hydrogen, theformula represents a “carboxylic acid”.

As used herein, “enzyme” can be any partially or wholly proteinaceousmolecule which carries out a chemical reaction in a catalytic manner.Such enzymes can be native enzymes, fusion enzymes, proenzymes,apoenzymes, denatured enzymes, farnesylated enzymes, ubiquitinatedenzymes, fatty acylated enzymes, gerangeranylated enzymes, GPI-linkedenzymes, lipid-linked enzymes, prenylated enzymes, naturally-occurringor artificially-generated mutant enzymes, enzymes with side chain orbackbone modifications, enzymes having leader sequences, and enzymescomplexed with non-proteinaceous material, such as proteoglycans,proteoliposomes. Enzymes can be made by any means, including naturalexpression, promoted expression, cloning, various solution-based andsolid-based peptide syntheses, and similar methods known to those ofskill in the art.

The term “C₁₋₆heteroaralkyl”, as used herein, refers to a C₁₋₆alkylgroup substituted with a heteroaryl group.

The terms “heteroaryl” includes substituted or unsubstituted aromatic 5-to 7-membered ring structures, more preferably 5- to 6-membered rings,whose ring structures include one to four heteroatoms. The term“heteroaryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings wherein at least one of the rings is heteroaromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, forexample, pyrrole, furan, thiophene, imidazole, isoxazole, oxazole,thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,phosphorus, and sulfur.

The terms “heterocyclyl” or “heterocyclic group” refer to substituted orunsubstituted non-aromatic 3- to 10-membered ring structures, morepreferably 3- to 7-membered rings, whose ring structures include one tofour heteroatoms. The term terms “heterocyclyl” or “heterocyclic group”also include polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings wherein atleast one of the rings is heterocyclic, e.g., the other cyclic rings canbe cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heterocyclyl groups include, for example,tetrahydrofuran, piperidine, piperazine, pyrrolidine, morpholine,lactones, lactams, and the like.

The term “C₁₋₆heterocycloalkyl”, as used herein, refers to a C₁₋₆alkylgroup substituted with a heterocyclyl group.

The term “C₁₋₆hydroxyalkyl” refers to a C₁₋₆alkyl group substituted witha hydroxy group.

As used herein, the term “inhibitor” is meant to describe a compoundthat blocks or reduces an activity of an enzyme (for example, inhibitionof proteolytic cleavage of standard fluorogenic peptide substrates,inhibition of various catalytic activities of the 20S proteasome). Aninhibitor can act with competitive, uncompetitive, or noncompetitiveinhibition. An inhibitor can bind reversibly or irreversibly, andtherefore the term includes compounds that are suicide substrates of anenzyme. An inhibitor can modify one or more sites on or near the activesite of the enzyme, or it can cause a conformational change elsewhere onthe enzyme.

As used herein, the term “peptide” includes not only standard amidelinkage with standard α-substituents, but commonly utilizedpeptidomimetics, other modified linkages, non-naturally occurring sidechains, and side chain modifications, as detailed below.

The terms “polycyclyl” or “polycyclic” refer to two or more rings (e.g.,cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Each of the rings of thepolycycle can be substituted or unsubstituted.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “prodrug” encompasses compounds that, under physiologicalconditions, are converted into therapeutically active agents. A commonmethod for making a prodrug is to include selected moieties that arehydrolyzed under physiological conditions to reveal the desiredmolecule. In other embodiments, the prodrug is converted by an enzymaticactivity of the host animal.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “proteasome” as used herein is meant to include immuno- andconstitutive proteasomes.

The term “repressing” is art-recognized, and when used in relation to acondition, such as cancer or any other medical condition, is wellunderstood in the art, and includes administration of a compositionwhich reduces the frequency of, or delays the onset of, symptoms of amedical condition in a subject relative to a subject which does notreceive the composition. Thus, repression of metastasis includes, forexample, reducing the number of detectable metastatic cancerous growthsin a population of patients receiving a prophylactic treatment relativeto an untreated control population, and/or delaying the appearance ofdetectable metastatic cancerous growths in a treated population versusan untreated control population, e.g., by a statistically and/orclinically significant amount.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include, for example, a halogen, ahydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl,or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, aphosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

The term “thioether” refers to an alkyl group, as defined above, havinga sulfur moiety attached thereto. In preferred embodiments, the“thioether” is represented by —S-alkyl. Representative thioether groupsinclude methylthio, ethylthio, and the like.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition.

Metastic Cancers

In accordance with the invention, a peptide epoxyketone or apharmaceutically acceptable salt thereof can be used in the repressionof metastasis of a cancer, including but not limited to hematologicalmalignancies, solid tumors, neuroblastoma, or melanoma.

In some embodiments of the invention, administration of the proteasomeinhibitor is initiated after a metastic tumor has been identified. Inother embodiments of the invention, administration of the proteasomeinhibitor is initiated after a primary cancer tumor has been identified.In certain preferred embodiments, administration of the proteasomeinhibitor is initiated after a primary tumor is identified, but prior tothe detection of a metastatic tumor. In certain embodiments of theinvention, the proteasome inhibitor is administered prophylactically toan individual susceptible to a metastatic cancer.

In certain embodiments, the cancer is a hematological cancer selectedfrom diffuse large B-cell lymphoma (DLBCL), T-cell lymphomas orleukemias (e.g., cutaneous T-cell lymphoma (CTCL), noncutaneousperipheral T-cell lymphoma, lymphoma associated with human T-celllymphotrophic virus (HTLV), and adult T-cell leukemia/lymphoma (ATLL)),acute lymphocytic leukemia, acute myelogenous leukemia (e.g., acutemonocytic leukemia and acute promyelocytic leukemia), chroniclymphocytic leukemia (e.g., chronic B cell leukemia), chronicmyelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma (e.g.,Burkitt's lymphoma), myeloma, multiple myeloma, and myelodysplasticsyndrome. In certain embodiments, the cancer is multiple myeloma. Incertain embodiments, the cancer is a lymphoma.

In certain embodiments the cancer is a solid tumor, neuroblastoma, ormelanoma selected from mesothelioma, brain neuroblastoma,retinoblastoma, glioma, Wilms' tumor, bone cancer and soft-tissuesarcomas, head and neck cancers (e.g., oral, laryngeal and esophageal),genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian,testicular, rectal, colorectal and colon), lung cancer (e.g., small cellcarcinoma and non-small cell lung carcinoma, including squamous cellcarcinoma and adenocarcinoma), breast cancer, pancreatic cancer, basalcell carcinoma, metastatic skin carcinoma, squamous cell carcinoma (bothulcerating and papillary type), stomach cancer, brain cancer, livercancer, adrenal cancer, kidney cancer, thyroid cancer, medullarycarcinoma, osteosarcoma, soft-tissue sarcoma, Ewing's sarcoma, reticulumcell sarcoma, and Kaposi's sarcoma. In certain embodiments, the canceris selected from breast cancer, cervical cancer, colorectal cancer,kidney cancer, lung cancer, melanoma, ovarian cancer (e.g., ovarianadenocarcinoma), pancreatic cancer and prostate cancer.

In certain embodiments, the cancer is selected from breast, cervical,colorectal, hematologic, kidney, lung, melanoma, neurological,pancreatic and prostate cancer.

Also included are pediatric forms of any of the cancers describedherein. This invention also provides a method for the treatment of drugresistant tumors. In certain embodiments, the drug resistant tumor ismultiple myeloma. In other embodiments, the drug resistant tumor is asolid tumor.

The term “drug resistant” as used herein refers to a condition whichdemonstrates intrinsic resistance or acquired resistance.

The term “intrinsic resistance” as used herein refers to thecharacteristic expression profile in cancer cells of key genes inrelevant pathways, including but not limited to apoptosis, cellprogression and DNA repair, which contributes to the more rapid growthability of cancerous cells when compared to their normal counterparts.

The term “acquired resistance” as used herein refers to a multifactorialphenomenon occurring in tumor formation and progression that caninfluence the sensitivity of cancer cells to a drug. Acquired resistancemay be due to several mechanisms such as but not limited to: alterationsin drug-targets, decreased drug accumulation, alteration ofintracellular drug distribution, reduced drug-target interaction,increased detoxification response, cell-cycle deregulation, increaseddamaged-DNA repair, and reduced apoptotic response. Several of thesemechanisms may occur simultaneously and/or may interact with each other.Their activation and/or inactivation can be due to genetic or epigeneticevents or to the presence of oncoviral proteins. Acquired resistance mayoccur to individual drugs but can also occur more broadly to manydifferent drugs with different chemical structures and differentmechanisms of action. This form of resistance is referred to asmultidrug resistance.

Administration of the Peptide Epoxyketone Proteasome Inhibitor

The peptide epoxyketone proteasome inhibitors as described herein can beadministered in various forms, depending on the disorder to be treatedand the age, condition, and body weight of the patient, as is well knownin the art. For example, where the compounds are to be administeredorally, they may be formulated as tablets, capsules, granules, powders,or syrups; or for parenteral administration, they may be formulated asinjections (intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. For application by the ophthalmic mucousmembrane route, they may be formulated as eye drops or eye ointments.These formulations can be prepared by conventional means, and ifdesired, the active ingredient may be mixed with any conventionaladditive or excipient, such as a binder, a disintegrating agent, alubricant, a corrigent, a solubilizing agent, a suspension aid, anemulsifying agent, a coating agent, a cyclodextrin, and/or a buffer.Although the dosage will vary depending on the symptoms, age and bodyweight of the patient, the nature and severity of the disorder to betreated or prevented, the route of administration and the form of thedrug, in general, a daily dosage of from 0.01 to 2000 mg of the compoundis recommended for an adult human patient, and this may be administeredin a single dose or in divided doses. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will generally be that amount of the compound which produces atherapeutic effect.

The precise time of administration and/or amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage, and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch, potatostarch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6)gelatin; (7) talc; (8) excipients, such as cocoa butter and suppositorywaxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such aspropylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol,and polyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations. In certainembodiments, pharmaceutical compositions of the present invention arenon-pyrogenic, i.e., do not induce significant temperature elevationswhen administered to a patient.

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, laurylsulphonate salts, and amino acidsalts, and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19.)

In other embodiments, the peptide epoxyketone proteasome inhibitorsuseful in the methods of the present invention may contain one or moreacidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic inorganic and organic base additionsalts of an inhibitor(s). These salts can likewise be prepared in situduring the final isolation and purification of the inhibitor(s), or byseparately reacting the purified inhibitor(s) in its free acid form witha suitable base, such as the hydroxide, carbonate, or bicarbonate of apharmaceutically acceptable metal cation, with ammonia, or with apharmaceutically acceptable organic primary, secondary, or tertiaryamine. Representative alkali or alkaline earth salts include thelithium, sodium, potassium, calcium, magnesium, and aluminum salts, andthe like. Representative organic amines useful for the formation of baseaddition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, and the like (see, forexample, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring, and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;(2) oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or nonaqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert matrix, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes, and the like, each containinga predetermined amount of an inhibitor(s) as an active ingredient. Acomposition may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), the active ingredient may bemixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, cyclodextrins, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols, andthe like. In certain embodiments, the crystalline tripeptide epoxyketoneis administered to a mammal as a capsule. In other embodiments, thecrystalline tripeptide epoxyketone is a compound of formula (I). In morepreferred embodiments, the crystalline tripeptide epoxyketone is acompound of formula (II).

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered inhibitor(s)moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills,and granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes, and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents, and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active inhibitor(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more inhibitor(s)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, which is solid at room temperature, butliquid at body temperature and, therefore, will melt in the rectum orvaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of aninhibitor(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams, and gels may contain, in addition toinhibitor(s), excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an inhibitor(s),excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The inhibitor(s) can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation, orsolid particles containing the composition. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular composition,but typically include nonionic surfactants (Tweens, Pluronics, sorbitanesters, lecithin, Cremophors), pharmaceutically acceptable co-solventssuch as polyethylene glycol, innocuous proteins like serum albumin,oleic acid, amino acids such as glycine, buffers, salts, sugars, orsugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of an inhibitor(s) to the body. Such dosage forms can be madeby dissolving or dispersing the agent in the proper medium. Absorptionenhancers can also be used to increase the flux of the inhibitor(s)across the skin. The rate of such flux can be controlled by eitherproviding a rate controlling membrane or dispersing the inhibitor(s) ina polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more inhibitors(s) in combination withone or more pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include tonicity-adjusting agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. For example, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microcapsule matrices ofinhibitor(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are, of course, given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection, and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug, or other materialother than directly into the central nervous system, such that it entersthe patient's system and thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These inhibitors(s) may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally, and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the inhibitor(s),which may be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present invention, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The concentration of a disclosed compound in a pharmaceuticallyacceptable mixture will vary depending on several factors, including thedosage of the compound to be administered, the pharmacokineticcharacteristics of the compound(s) employed, and the route ofadministration. In general, the compositions of this invention may beprovided in an aqueous solution containing about 0.1-10% w/v of acompound disclosed herein, among other substances, for parenteraladministration. Typical dose ranges are from about 0.01 to about 50mg/kg of body weight per day, given in 1-4 divided doses. Each divideddose may contain the same or different compounds of the invention. Thedosage will be an effective amount depending on several factorsincluding the overall health of a patient, and the formulation and routeof administration of the selected compound(s).

Combination Therapy

One aspect of the invention relates to the treatment of metastaticcancer, wherein a peptide epoxyketone or a pharmaceutically acceptablesalt thereof is administered with one or more other therapeutic agents.Such combination treatment may be achieved by way of the simultaneous,sequential, or separate dosing of the individual components of thetreatment.

In certain embodiments, the one or more other therapeutic agent isselected from an HDAC inhibitor, an antibiotic, a taxane, anantiproliferative/antimitotic alkylating agents, a platinum coordinationcomplex, a steroid, an immunomodulator, a topoisomerase inhibitor, anm-TOR inhibitor, protein kinase inhibitor, another proteasome inhibitoror radiotherapy.

In certain embodiments, the other therapeutic agent is an HDAC inhibitor(e.g., Trichostatin A, depsipeptide, apicidin, A-161906, scriptaid,PXD-101, CHAP, butyric acid, depudecin, oxamflatin, phenylbutyrate,valproic acid, SAHA (Vorinostat), MS275(N-(2-Aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide),LAQ824/LBH589, CI994, and MGCD0103). In certain such embodiments, theother agent is SAHA (suberoylanilide hydroxamic acid).

In certain embodiments, the other therapeutic agent is an antibiotic(e.g., dactinomycin (actinomycin D), daunorubicin, doxorubicin andidarubicin). In certain such embodiments, the other therapeutic agentcomprises doxorubicin. In certain such embodiments, the othertherapeutic agent is Doxil.

In certain embodiments, the other therapeutic agent is a taxane (e.g.,paclitaxel and docetaxel).

In certain embodiments, the other therapeutic agent is anantiproliferative/antimitotic alkylating agents such as a nitrogenmustard (e.g., mechlorethamine, ifosphamide, cyclophosphamide andanalogs, melphalan, and chlorambucil). In certain such embodiments, theother therapeutic agent is cyclophosphamide or melphalan.

In certain embodiments, the other therapeutic agent is a platinumcoordination complex (e.g., cisplatin and carboplatin). In certain suchembodiments, the other therapeutic agent is carboplatin.

In certain embodiments, the other therapeutic agent is a steroid (e.g.,hydrocortisone, dexamethasone, methylprednisolone and prednisolone). Incertain such embodiments, the other therapeutic agent is dexamethasone.

In certain embodiments, the other therapeutic agent is animmunomodulator (e.g., thalidomide, CC-4047 (Actimid), and lenalidomide(Revlimid). In certain such embodiments, the other therapeutic agent islenalidomide.

In certain embodiments, the other therapeutic agent is a topoisomeraseinhibitor (e.g., irinotecan, topotecan, camptothecin, lamellarin D, andetoposide).

In certain embodiments, the other therapeutic agent is an m-TORinhibitor (e.g., CCI-779, AP23573 and RAD-001).

In certain embodiments, the other therapeutic agent is a protein kinaseinhibitor (e.g., sorafenib, imatinib, dasatinib, sunitinib, pazopanib,and nilotinib). In certain such embodiments, the protein kinaseinhibitor is sorafenib.

Administration of the peptide epoxyketone may precede or follow theother therapeutic agent by intervals ranging from minutes to days. Incertain such embodiments, the peptide epoxyketone and the othertherapeutic agent may be administered within about 1 minute, about 5minutes, about 10 minutes, about 30 minutes, about 60 minutes, about 2hours, about 4 hours, about 6 hours, 8 hours, about 10 hours, about 12hours, about 18 hours, about 24 hours, about 36 hours, or even about 48hours or more of one another. Preferably, administration of the peptideepoxyketone and the other therapeutic agent will be within about 1minute, about 5 minutes, about 30 minutes, or even about 60 minutes ofone another.

In certain embodiments, the peptide epoxyketone and the othertherapeutic agent may be administered according to different dosingschedules (e.g., the peptide epoxyketone, for example may beadministered once a day while the other therapeutic agent may beadministered only once every three weeks) such that in some instancesadministration of the peptide epoxyketone and the other therapeuticagent will be within about 60 minutes of one another, while in otherinstances, administration of the peptide epoxyketone and the othertherapeutic agent will be within days or even weeks of one another.

As used herein, the term “regimen” is a predetermined schedule of one ormore therapeutic agents for the treatment of a cancer. Accordingly, whena therapeutic agent is administered “alone,” the regimen does notinclude the use of another therapeutic agent for the treatment ofcancer.

In certain embodiments, combinations as described herein may besynergistic in nature, meaning that the therapeutic effect of thecombination of the peptide epoxyketone and the other therapeuticagent(s) is greater than the sum of the individual effects.

In certain embodiments, combinations as described herein may be additivein nature, meaning that the therapeutic effect of the combination of thepeptide epoxyketone and the other therapeutic agent(s) is greater thanthe effect of each agent individually (i.e., the therapeutic effect isthe sum of the individual effects).

EXEMPLIFICATION Example 1

6-8 week old female BALB/c mice were challenged in the mammary fat padwith 4T1 mammary carcinoma cells (1×10⁵/mouse). Treatment was initiatedon Day 3 post tumor challenge. Compound 1 was administered orally at 30or 40 mg/kg on a weekly schedule of QDX2 or QDX5.

Compound 1 treatment reduced the number of metastatic tumors in the lungby about 50%. Both dose schedules and dose levels of Compound 1 testedwere effective in repressing metastasis (FIG. 1).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims.

All of the above-cited references and publications are herebyincorporated by reference.

We claim:
 1. A method for repressing metastasis of a cancer, comprisingadministering a therapeutically effective amount of a peptideepoxyketone proteasome inhibitor or a pharmaceutically acceptable saltthereof.
 2. The method of claim 1, wherein the peptide epoxyketone has astructure of formula (I) or a pharmaceutically acceptable salt thereof

wherein each Ar is independently an aromatic or hetero aromatic groupoptionally substituted with 1-4 substituents; L is selected from C═O,C═S, and SO₂; X is selected from O, S, NH, and N—C₁₋₆alkyl; Y is absentor is selected from C═O and SO₂; Z is absent or is C₁₋₆alkyl; R¹, R²,and R³ are each independently selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl,C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, any of which is optionallysubstituted; R⁴ is N(R⁵)L-Z—R⁶; R⁵ is selected from hydrogen, OH,C₁₋₆aralkyl-Y—, and C₁₋₆alkyl-Y—; R⁶ is selected from hydrogen, OR⁷,C₁₋₆alkenyl, Ar—Y—, carbocyclyl, and heterocyclyl; and R⁷ and R⁸ areindependently selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl.
 3. Themethod of claim 1, wherein the peptide epoxyketone has a structure ofFormula (II) or a pharmaceutically acceptable salt thereof

wherein L is selected from C═O, C═S, and SO₂; X is O; Z is absent,C₁₋₆alkyl, or C₁₋₆alkoxy; R¹, R², and R³ are each independently selectedfrom hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆hydroxyalkyl,C₁₋₆alkoxyalkyl, aryl, C₁₋₆aralkyl, heteroaryl, heterocyclyl,C₁₋₆heterocycloalkyl, C₁₋₆heteroaralkyl, carbocyclyl, andC₁₋₆carbocyclolalkyl; R⁴ is selected from hydrogen, C₁₋₆aralkyl, andC₁₋₆alkyl; R⁵ is heteroaryl; and R⁶ and R⁷ are independently selectedfrom hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl.
 4. The method of claim 3,wherein the peptide epoxyketone has the following structure:

or a pharmaceutically acceptable salt thereof.
 5. The method of claims1-4, wherein the cancer is breast, cervical, colorectal, hematologic,kidney, lung, melanoma, neurological, pancreatic or prostate.
 6. Themethod of claims 1-5, wherein the peptide epoxyketone is administeredorally.
 7. The method of claim 6, further comprising administering oneor more additional therapeutic agents.
 8. A method of treating anindividual who has been identified as being susceptible to metastasizedcancer, comprising administering to the individual a prophylacticallyeffective amount of a peptide epoxyketone proteasome inhibitor.